Background of Cell-Free Protein Synthesis

Since the early pioneering work of Nirenberg and Matthaei in 1961 (1), which demonstrated in vitro protein translation using cell extracts, cell-free protein synthesis has become an important tool for molecular biologists by playing a central role in a wide variety of applications (2). In the post-genomic era, cell-free protein synthesis has the potential to become one of the most important high throughput technologies for functional genomics and proteomics.

The biggest advantage, compared to protein production in living cells, is that cell-free protein synthesis is the quickest way to obtain an expressed phenotype (protein) from a genotype (gene). Starting with a PCR or plasmid template, in vitro protein synthesis and functional assays can be carried out in a few hours. Moreover, it is independent of host cells. Proteins which are toxic or prone to proteolytic degradation can be readily prepared in vitro.

Commercially available cell-free protein synthesis systems are typically derived from cell extracts of Escherichia coli S30, rabbit reticulocytes or wheat germ. The drawback of extract-based systems is that they often contain nonspecific nucleases and proteases that adversely affect protein synthesis. In addition, the cell extract is like a “black box” in which numerous uncharacterized activities may modify or interfere with subsequent downstream assays.

Some of these limitations can be partially overcome, for instance, by using engineered strains or by adding various inhibitors. Nevertheless, the problems cannot be solved at the root level.